Projects / Programmes
Electrostatic immobilisation of bacterial cells and effects on their physiology
| Code |
Science |
Field |
Subfield |
| 4.06.04 |
Biotechnical sciences |
Biotechnology |
Microbe biotechnology |
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.08 |
Engineering and Technology |
Environmental biotechnology
|
electrostatic immobilization of bacteria, bacterial encapsulation, molecular microbiology, colloid physics
Researchers (18)
Organisations (5)
Abstract
In nature, the extracellular matrix, which encloses bacterial cells, has a vital role for normal cell functioning. Such confinement serves as protection against predators and antimicrobial agents, allows the establishment of high cell densities and co-aggregation of different types of cells and provides a matrix, where different bioactive molecules can concentrate. In biotechnological processes these natural principles can by exploited by artificially implementing immobilization of bacterial cells. It has been shown in several cases that this approach can be extremely potent, due to procedure simplification and increased efficiency of the biotechnological processes. However, currently established methods of cell immobilization are generally based on aggregation of bacterial cells by dehydration and consecutive pelletisation, by entrapment in gel or sol-gel based matrix or within a cross-linked natural (e.g. alginate, carrageenan) or artificial polymer (e.g. polyacrylamide, polyethylene glycol, polyvinyl alcohols) based matrix.
Since the matrix-cell interaction is based on physicochemical principles, the knowledge of colloid physics can be extremely important for the development of new immobilization strategies. One such new strategy can be based on the adaptation of deposition of charged polymers used in layer-by-layer (LBL) methods. This method has been extensively investigated and fully exploited for surface modifications of inorganic and organic nonliving materials, either on two dimensional (surfaces of different materials) or on three dimensional surfaces (micro-, nano-particles). Capsules that are formed this way, are very thin, as well as porous, but at the same time have tremendous elastic and strength properties, since polymers are deposited on a surface in thin tailor made shell, where each layer is directly deposited on charged surface forming a mesh of a few nm thickness. By utilizing different LBL encapsulation strategies through variation of ionic strength, pH, different types and sizes of polymers and levels of branching, as well as induction of hydrogen and covalent bonds between polymers, the properties, such as porosity, capsule thickness and strength as well as surface charge, can be controlled. Since bacterial cells can be treated as a special case of soft matter, with the membrane and cell wall surface forming a negatively charged surface, we can exploit this intrinsic property for encapsulation, surface modification, entrapment and immobilization, by using polyelectrolyte polymers in a similar way as deposition of polyelectrolytes on non-living surfaces or particles using modified conventional LBL method.
Accordingly, our hypothesis is that it is possible to permanently and efficiently entrap bacterial cells within layers of polyelectrolytes. Contrary to past studies, we are taking into account that micron sized bacterial cells are different than non-living micro-particles normally used in LBL encapsulation, since these cells: (i) have very diverse surface softness due to production of extracellular matrix of different texture, as well as dynamic wall architecture, (ii) can adapt metabolically or by cell surface organization after the deposition of charged colloids on cell surface, (iii) positively charged polycations can affect viability of bacterial cells and most notably (iv) LBL coating of cells affects cell physiology due to capsule porosity, diffusion of nutrients and by physical mitigation of cell mass gaining and division. Based on this hypothesis the aims in this project are to: (i) develop an LBL immobilization strategy for bacterial cells, (ii) characterize physicochemically and microscopically such LBL immobilized bacteria, (iii) determine the effect of polyelectrolyte encapsulation on physiology and cell division and (iv) to evaluate the changes of mass balances of LBL immobilized cells in comparison to free-living cells.
Significance for science
In biotechnology it is recognized the importance of immobilisation of cells and many important effects has been described. For example, it is known that encapsulation prevents competition with other cells and destruction of bacteria important in biotechnological processes by grazing with protozoans. Encapsulation can increase production of substances in biotechnological processes (e.g. production of ethanol from yeast cells). However, currently in most cases the immobilisation techniques are based on the entrapment into the polymer matrix like alginate.
In recent years the methods of use of polyelectrolytes has been also applied on the cell. However, although this method showed many promising results, it was not considered as depositing polyelectrolytes on the surface of cells, but more like depositing polyelectrolytes on the surface of nonliving particles and most of the important and essential questions remained unanswered. Since, in the research within this project we are going to observe interaction of cells with charged polyelectrolytes, there are at least four important major advancements in the scientific field:
i) we will obtain insights of mechanophysical interaction of charged colloids with bacterial surfaces,
ii) we will obtain physiological insights of either effects of attachment of charged colloids on cell surface or mechanical mitigation of encapsulated cells within LbL formed capsules,
iii) we will determine the factors affecting formation of aggregates and layers of cells and we will determine how this spatial forms of cells effect on their physiology,
iv) we will obtain in deep understanding effect of spatial distribution of different bacterial cells on their interaction between each others
These advancements in the field will enable deep understanding of functioning of bacterial cells in entrapped environment such as small pores in soil and will enable development of different methods for bacterial cell surface modifications. Here, surface modifications of bacterial cells enable investigation of interaction of bacterial cells in the environment, since the new developed methods within the project as well as determination of their limitations, will enable spatial investigations of interactions of one cell with other sorts of cells. It can also enable investigation of biofilm formation by placing cells in a particular space. The changes in the structure of the deposited cells can then be monitored also over particular time window. This is currently hot topic in the investigation of the ecology of attached bacteria and their spatial distribution.
The relevance of the development of the scientific field is based on our current knowledge and on our overview of the state-of-the-art of published articles, shown in the attachment Literature and in C.13
Significance for the country
Immobilisation strategies of bacterial cells simplify and increase the efficiency of biotechnological processes. They not only reduce costs but also reduce the environmental footprint. The project disrupts the biotechnology sector with the development of novel LbL approach of electrostatic modification of bacterial cell surfaces that will enable the use of living organisms in advanced GMO free biotechnological applications.
The project aims to increase the performance of biotechnological processes and applications while securing the sustainable development, consequently impacting mostly the health care, agricultural, industrial and environmental biotechnology sectors. It is aligned with concepts of green growth (OECD)2, the green economy (UNEP)3 and a materially-efficient and low carbon society4 (EC), which are based on improving the energy, material, environmental, and social efficiency, and complies with the objectives of Horizon 2020 and European development policy.
The project will enable the advancement in the fields of: (i) tissue engineering, (ii) microencapsulation for diseases treatment, (iii) food and beverage applications, and (iv) industrial biochemical production. The consortium is interdisciplinary with partners coming from the research, educational and business sector, what ensures us to make heavy impact on the biotech sector through already established connections with the global academic and industrial sector. Therefore project complies with the Smart Specialisation Strategy of the Republic of Slovenia outlining the importance of cooperation of different stakeholders to foster knowledge transfer and internationalisation. The project results offer a possibility for patent protection and grounds for further applicative development of new products and services at already established companies, and enable the evolvement of new dedicated “spin-off” companies, promoting competitiveness, and the social and economic empowerment of slovenian citizens.
The project will enable to create and spread new ties with complementary organisations, and will despite being a basic scientific project, foster a translation of the basic knowledge at using electrostatic modification of bacterial cell surfaces into applicative solutions. The project will open culture that would allow for free exchange of ideas, share and compare experience on selected topics throughout education of young researchers and transfer knowledge between partners and to students.
OECD, The Application of Biotechnology to Industrial Sustainability - A Primer: http://www.oecd.org/sti/biotech/1947629.pdf
OECD (2011), Towards the Green Growth, OECD Publishing.
United Nations Environment Programme, Green Economy: http://www.unep.org.
European Commission, Low Carbon Society: http://ec.europa.eu/clima/citizens/aworldyoulike/index_en.htm
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
